CMMB 411- Topic 3 Memory

DNA Pol III Core consists of: (PROK)

alpha: synthesis

epsilon: 3’-5’ proofreading

theta: non-essential

DNA Holoenzyme consists of (PROK):

alpha, epsilon, and theta +

3 beta-sliding clamps

1 clamp loader

psi and phi

Clamp Loader consists of (PROK):

3 T subunits - C term linker, bind DnaB and Pol III Core

delta: open clamp

delta’: modulate delta

***remember this is apart of Pol III holoenzyme

Sliding Clamp consists of (PROK):

Homodimer of 6 units

Function of beta-sliding clamp

Increase processivity

How is the clamp loaded? (PROK)

-ATP binds T subunits

- change in conformation (open sliding clamp)

- bind DNA and the primer junction

- ATP hydrolosis

• Cause loader to open and leave DNA

Helicase (PROK):

Structure too

DnaB

homohexamer

Binds to T subunit

ATP!!!

ENTIRE DNA Pol III Holoenzyme + Helicase- connections, and all subunits

3 DNA Pol III Cores

• Alpha, epsilon, theta

1 Clamp Loader

• 3 T subunits - bind to 

• Delta, delta’

3 Sliding Clamp

• Homohexamer of 6 subunits, binds to DNA Pol III through T subunit

Cai and PSI - keeps loader to Pol III

DnaB helicase- bound to Pol III through T subunits

Topioisomerase:

Decrease Lk

Relax + supercoil

TopoIII

Primase (PROK):

DnaG

SSB Protein (PROK):

Produced by helicase

Cooperative

Protect ssDNA (no secondary structures!)

Prokaryotic Replisom:

DNA Pol III

DnaB - helicase

DnaG - primase

Replisome Progression Complex (EUK):

Ctf4

Mcm10

(CMG)

• Mcm2-7

  Cdc45

  GIN

Eukaryotic Helicase:

Mcm2-7

Hexameric, ringed

Eukarytoic Clamp:

PCNA

Eukaryotic Clamp Loader:

RFC

Eukaryotic SSB Protein:

RPA

CMG Consists of: ____, _____, _____

It is a _____ ______ complex

It travels on the _____ strand

Cdc45, Mcm2-7, GINS

progressive helicase

leading 

Eukaryotic Primase:

DNA Pol alpha

Has RNA and DNA polymerase activity

Replicative DNA polymerases (EUK):

They carry out ____ translation with ______

DNA Pol Epsilon: synthesize leading strand

DNA Pol Delta: synthesize lagging strand

nick translation, FEN 1 Flap Endonuclease

Fast Stop

DNA synthesis protein mutation

Slow Stop

DNA initiation protein mutation

3 Mechanisms for preventing re-anealing in prok

1. Methylation Mechanism by DAM

2. DnaA-ATP dependent

3. RNA Pol Dependent

Methylation Mechanism- proteins and function?

DAM- recognize GATC sequence and methylate A on parent

SeqA- sees methylation, and binds to the hemimethylated strands

DnaA-ATP Mechanism - proteins and function?

DnaA requires ATP to function

HdA hydrolizes ATP on DnaA

DnaA sequesters to another 9mer spot

Slow ATP kinetics -no reinitation

RNA Pol Dependent Mechanism- proteins and function?

RNA Pol transcribing a gene proximal to OriC = A-T region opens

DnaA cant bind to it

Restriction Point

G1/S Checkpoint

Commit to complete a round of division *if condn is correct

Cdk2-Cyclin E

Entry into the S phase

Phosphorylate E2F (TF)

activate DNA replication genes

Oncogenes

Positive regulation

Gain of Function= Cancer

Cyclin D/E

Cdk4

Tumour Supressor Genes

Negative regulation

Loss of function= Cancer

p53, ChK2, p16, RB

Eukarytoic pre-Initation

ORC binds to ______

ARS at B1 and A

Cdc6 and Cdt4 bring _____ to ______

This makes the ____

2 helicases (Mcm2-7) to B1 and A

pre-initation complex

CDK/DDK ______ substrates and ____

phosphorylate the substrates of the pre-RC and recruit CMG

Liscencing Factors

Cdc6, Cdt4 = after origin firing, degraded by CDK

G1: CDK, pre-RC formation, pre-RC activity

G1: CDK low, pre-RC form high, pre-RC act. low

S: CDK, pre-RC formation, pre-RC activity

S: CDK high, pre-RC form low, pre-RC act. high

Prokaryotes invole _____sites for _____ termination

TER sites, replication

Bind to TER sites

TUS proteins

TUS proteins are…..

unidirectional

TER sites prevent (2 things)

Head on collision, replication of genome once

Telomerase adds:

Noncoding ssDNA to 3’end of chromosome

Human Telomere sequence:

5’-TTAGGG-3’

Linearlized yeast plasmid with tetrahymena telomere sequence could..

maintain and add telomeres, and were YEAST telomeres

Telomerase Subunits

TERT, RNA Subunit

TERT

Reverse Transcriptase 

RNA Subunit

Telomerase RNA/TR- template for telomere synthesis

Tetrahymena TR sequence:

CAACCCCAA

Telomere repeat sequence

GGTTG

Shelterin proteins

TRF1, TFR2

POT1

TIN2, TPP1

RAP1

What does shelterin do?

Control telomere length-influence telomerase activity

TRF1 and TRF2:

DIRECT contact with DNA

POT1: -3 things

Bind 3’ ssDNA overhang
Control telomere length

Inhibit telomerase activity -prevent tr acsess

TIN2 and TPP1:

keep complex intact

RAP1:

inhibit non-homologous end joining

Shorter telomere: ____ POT1 = _____telomerase

less, more

Why does the overhang need to be “controlled”?

Think it is a DSB- will try NHEJ

T Loop formation

ssDNA 3’ overhang folded back
form dsDNA loop - via strand invasion

T loops are important becasue:

Hide end of telomere, not broken chromosome